The objectives of this pilot and feasibility project are to develop a method to study the glucose transport system in human skeletal muscle and then to use this method to investigate a potential mechanism of the peripheral insulin resistance that is associated with obesity. Previous studies of the glucose transport system in human skeletal muscle have revealed that the expression of glucose transporter proteins is not significantly altered in obese patients and therefore, can not explain the characteristic decrease in insulin-stimulated glucose uptake associated with this condition. The current study is designed to determine if the functional components of the glucose transport system, such as the subcellular distribution of glucose transporter proteins, insulin- stimulated translocation of glucose transporters, and the intrinsic activity of glucose transporters, are altered in the skeletal muscle of obese patients. For this purpose, a procedure to isolate subcellular membrane fractions from human skeletal muscle will be adapted from techniques originally developed in rat skeletal muscle. The isolated plasma and microsomal membranes will then be used to study; 1.) the expression of the GLUT-1, GLUT-3, GLUT-4, and GLUT-5 transporter isoforms by western blot procedures; 2.) the subcellular distribution of each isoform and of all glucose transporter proteins by the cytochalasin B binding technique; and 3.) insulin-stimulated translocation of glucose transporter proteins. In addition, glucose transport in plasma membrane vesicles will be measured, the transporter turnover number calculated, and the intrinsic activity of the glucose transporter proteins determined. These studies will be performed before and after an insulin clamp procedure. Glucose transporter functional measurements (i.e. translocation, vesicle transport, intrinsic activity) will be correlated with in vivo measurements (total glucose disposal, glucose oxidation, non-oxidative glucose disposal, endogenous glucose production). These studies will be performed in healthy volunteers and obese insulin resistant patients before and after diet therapy. These studies will provide critical information for determining the cellular mechanism of peripheral insulin resistance associated with obesity.